Carlos Lois - US grants

[unreadable] DESCRIPTION (provided by applicant): The human brain has a limited ability for repair, and thus, diseases or injuries to the nervous system are usually irreversible. The addition of new neurons into the olfactory bulb and hippocampus of the adult mammalian brain suggests that cell replacement may be a promising strategy for brain repair. However, developing successful strategies for cell-replacement based brain repair requires an understanding of how newly generated neurons integrate into pre-existing, functioning neural circuits in the adult brain. Previous observations suggest that the incorporation of new neurons into the olfactory bulb of newborn animals is more efficient than in adult animals. The goal of this proposal is to understand the mechanisms that may regulate the increased ability of new neurons generated in the newborn brain to integrate into the olfactory bulb, compared to adult-born neurons, with the long-term objective of applying this understanding towards improving strategies for brain repair. An important clue comes from our recent discovery that new neurons added to the olfactory bulb in newborn rodents integrate into the bulb circuit with a pattern of connectivity distinct from that of the adult animal. This difference offers an ideal experimental opportunity with which to uncover the mechanisms that regulate how new neurons integrate into functioning brain circuits. In this proposal we will concentrate our efforts on three aims. First, we will investigate the differences in the connectivity of interneurons generated in the adult versus the newborn olfactory bulb by labeling neuronal progenitors with retroviruses to analyze their morphology, and will use a genetic marker to reveal their synaptic input. Second, we will assay the contribution of cell-autonomous or target-dependent mechanisms in the establishment of the different patterns of connectivity of new neurons born in postnatal olfactory bulb by analyzing the morphology of granule neurons derived from neuronal progenitors after transplantation into isochronic or heterochronic hosts. Third, we will characterize how genetic manipulations of activity affect the integration of new neurons born in the bulb of newborns or adults by delivering into neural progenitors three different constructs to alter the intrinsic excitability and the synaptic properties of newly generated bulb neurons. This proposal will investigate how different classes of neurons survive and connect to other neurons in the brain of adult and newborn animals. The insights gained from the experiments that we describe will guide future efforts towards harnessing replacement of neurons as a strategy for brain repair in humans. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Songbirds have been a rich experimental system for studying neurobiological questions of relevance to human health for decades. In particular, songbirds are the leading non-human model for the investigation of the biological basis of vocal learning, a critical behavioral substrate for speech acquisition. In addition, songbirds are an ideal system for the study of brain asymmetry, hormonal control of brain development, physiological function of sleep, and sex-specific differences in the brain, behavioral-induced gene expression and adult neurogenesis, among other questions. Nonetheless despite their importance for neurobiology, the usefulness of songbirds as an experimental system has been restricted by a lack of genetic manipulation methods. Recently we have succeeded in applying lentiviral-mediated transgenesis to zebra finches (Agate et al., 2009). While this achievement proves the feasibility of lentiviral-mediated transgenesis in songbirds, the current method is inefficient, labor-intensive and costly. In this proposal we plan to optimize the existing strategy and to generate novel tools to develop a practical avian transgenic technique that could be used by other songbird neurobiology laboratories. The ability to perform genetic manipulations in songbirds will open unparalleled opportunities for the study of the relationship between genes and brain function in an animal species with a robust behavioral repertoire. Transgenic songbirds will allow us to generate animal models of human diseases affecting complex cognitive functions and communication disorders, which can only be incompletely modeled in other animal species. PUBLIC HEALTH RELEVANCE: The main manifestation of many neurological and psychiatric disorders in humans is altered behavior. The rich behavioral repertoires exhibited by songbirds make them ideal animal's nriodels in which to mimic human diseases that affect behavior. We propose to develop new molecular tools that will allow for genetic manipulation of songbirds to generate animal models of human diseases that affect complex cognitive functions and communication disorders.

DESCRIPTION (provided by applicant): Communication impairments are some of the most debilitating consequences of a number of neurodevelopmental disorders, including autism spectrum disorders (ASD). It is thought that the communication impairments associated with neurodevelopmental disorders may be due to the perturbed assembly of brain areas involved in vocal communication. In recent years a number of genes, including the transcription factor FoxP2 and its target CNTNAP2, have been linked to language specific impairments and to ASD in humans, suggesting that they could play a direct role in speech and language acquisition. However, the lack of adequate animal models to specifically study vocal learning and communication has prevented the dissection of the genetic components involved in communication behaviors. Songbirds are currently the best animal model to study vocal communication because song learning in these animals shares critical features with speech acquisition in humans. Moreover, songbirds have a dedicated brain circuit that is required for the learning and production of vocal signals. Finally, both Foxp2 and CNTNAP2 are expressed in the song system suggesting that speech acquisition in humans and song learning in songbirds involves similar molecular pathways. Thus, songbirds could be an excellent model to study the genetic basis of communication-related aspects of neurodevelopmental disorders, as they represent the ideal system to study how genes orchestrate the assembly of a brain circuit dedicated to vocal learning and production. Our laboratory has developed new methods for the genetic modification of songbirds that will enable us and other researchers to generate genetic animal models for vocal communication disorders. In this proposal we describe a plan to generate genetically-engineered songbirds deficient in CNTNAP2, which will open a new avenue for investigating the role of genes on the assembly and function of the brain circuits involved in vocal communication. This approach should lead to a better understanding on how mutations in this and other genes result in communication deficits such as the ones observed in patients with ASD and other neurodevelopmental disorders.

DESCRIPTION (provided by applicant): Understanding the computations that take place in brain circuits requires identifying how neurons in those circuits are connected to each other. In recent years several new approaches (most notably, serial electron microscopy, replication-deficient rabies viruses, and GRASP) have been designed to identify the wiring diagrams of brain circuits. To overcome some of the limitations of the currently available strategies we propose to generate a new genetically-encoded system to trace brain circuits by transsynaptic control of transcription that will open new opportunities for investigating the relationship betwee circuit connectivity and function. The system that we propose is based on the molecular logic of the Notch receptor. In this system, neurons expressing an artificial ligand (sender neurons) activate a genetically-modified Notch receptor on their synaptic partners (receiver neurons). Upon ligand-receptor interaction in synaptic sites, the engineered receptor is cleaved in its transmembrane domain and releases a protein fragment that regulates transcription in the synaptic partners. Our initial experiments in vitro have confirmed the feasibility of this strategy and we propose to apply this design towards identifying wiring diagrams of neuronal circuits in transgenic animals, both in mice and drosophila.

? DESCRIPTION (provided by applicant): Recent genetic advances have driven significant progress in scientists' abilities to classify and map neuronal cell types within the brains of mode organisms like laboratory mice. To better delineate neuronal cell types in the human brain, however, it is critical to have a deeper understanding of the way that neuronal cell types evolve across mammals. As a first step toward achieving this understanding, corresponding neuronal cell types will be directly compared in two closely related mammalian species: mice and rats. By closely examining differences in the properties of these cells, including the genes they express, we hope to identify genomic elements that control the properties of neuronal cell types, and to infer properties of the corresponding cell types in the human brain. Improving the precision with which we can classify human neuronal cell types could have a transformative impact on our ability to understand pathological changes in neuropsychiatric disease.

? DESCRIPTION (provided by applicant): The neural mechanisms underlying learned motor behaviors are one of the least understood aspects of neuroscience, yet treatment of motor deficits after injury or disease is a significant clinical need. A better understanding of the organization of motor control could lead to effective treatments that provide enhancements of motor plasticity, or brain machine interfaces for functional restoration. However, at the cellular level, little is known about how brain circuits are able to learn and reproduce temporal sequences. One the greatest challenges involved in investigating natural motor control is the lack of repeatability of the learned motor skill. To image neuronal activity during the execution o a highly complex, yet stereotyped, behavior we propose to develop a transgenic songbird expressing the genetically-encoded Ca++ indicator GCaMP6. We will use the zebra finch, a species that learns to produce an extremely stereotyped song pattern, with strikingly small trial-to trial variations. Thus, this system provides a rare model from which to tightly correlate functional changes in neuronal activity to specific and readily measurable behavioral changes. Uncovering principles relating motor behavior and cell type specific circuit activity will provide key insights governing sensory-motor learning, adaptive plasticity in response to injury, and also provide insights for the development of next generation brain-machine interfaces.

? DESCRIPTION (provided by applicant): Understanding the computations that take place in brain circuits will require identifying the wiring diagrams of those circuits. In recent years seveal new methods have been developed to identify the brain's wiring diagrams. Each of these methods have some strengths and limitations. Importantly, there is no available anterograde monosynaptic tracer that can be used to regulate gene expression of synaptically connected neurons in species ranging from drosophila to mice. We propose to develop and validate a new genetically-encoded system to trace brain circuits by transsynaptic control of transcription that could overcome some of the limitations of the currently available strategies. We anticipate that this tool will open new opportunities for investigating the relationship between connectivity of neuronal circuits and brain function. The strategy that we propose is based on ligand- induced intramembrane proteolysis. In this system, neurons expressing an artificial ligand (emitter neurons) activate an engineered receptor on their synaptic partners (receiver neurons). Upon ligand-receptor interaction in synaptic sites, the engineered receptor is cleaved in its transmembrane domain and releases a protein fragment that regulates transcription in the synaptic partners. Our initial experiments in vivo, in transgenic drosophila, have confirmed the feasibility of this strategy as a method to record cell-cell interactions between neurons in the brain. We propose to optimize and validate this design towards identifying wiring diagrams of neuronal circuits in transgenic animals, both in mice and drosophila.

Over the past four decades, advances in gene manipulation technologies have dramatically improved our understanding of numerous fields in biology. However, although studies in birds have made seminal contributions to fields such as development, neurobiology, and immunology, bird research has been hindered by the limited availability of gene manipulation tools, including the ability to make transgenic birds. In other model species, such as mice, zebrafish, and fruit flies, it is much easier to generate transgenic animals that directly assess the role of a particular gene of interest. Some of the most successful transgenic technologies rely on gene-editing and isolating and modifying stem cells, and then transplanting them into hosts. In contrast, in birds, the advanced age of embryos at the time eggs are laid, and the lack of efficient viral vector tools has greatly limited gene manipulation efforts. To address these limitations, the investigators develop and improve gene manipulation and stem cell technologies in birds. The project focuses on the zebra finch, a vocal learning songbird that is the most commonly used animal model to study the neural and genetic basis of human speech and language. Since other animal models commonly used in research do not have vocal learning, this is the first time that efficient methods for gene manipulation are being developed for a vocal learner species. Other beneficiaries include all avian research and possibly any egg-laying species, many of which are used to address questions in diverse areas of biology. The project also provides research opportunities for high school, undergraduate, and graduate students and outreach to communities typically underrepresented in science and technology fields.

To provide gene manipulation tools and protocols that will be useful not only to the songbird research community, but also to avian researchers in general, the investigators are: 1) generating transgenic zebra finches that express the genome-editing enzyme Cas9 under a ubiquitous promoter; 2) isolating, culturing, and utilizing primordial germ cells (PGCs) to improve the efficiency of generating transgenic zebra finches; and 3) developing efficient viral vectors for manipulating genes in zebra finch cells. Using established methods transgenic zebra finches are made by injecting VSV-pseudotyped lentiviral vectors into freshly-laid fertilized eggs. When combined with CRISPR-designed guide RNAs, these transgenic songbirds enable gene-editing capabilities in a variety of tissues and cell types. To scale up and create many transgenic lines, PGC culture methods are being optimized and viral vectors with higher transfection rates in zebra finch cells developed, thus reducing the materials, time, and animals needed to create a new transgenic line, as well as greatly facilitating gene manipulations. The tools and optimized methods generated by this project will also impact avian research in fields other than birdsong biology. PGC culturing protocols can be applied to other avian species, viral vectors optimized for specific songbird tissues may also have higher transfection rates in analogous cell types of other avian species, and the Cas9-lentiviral construct could be used to generate other strains of Cas9-expressing transgenic birds.

Abstract How do the cell fates, lineages, and molecular histories of individual cells control the development and physiological function of tissues? This problem is of central importance throughout biomedical science. However, progress has long been limited by two seemingly intractable, and inter-related, challenges: First, we still have no method to determine the tree of lineage relationships among cells in developing tissues within their native spatial context. Second, we have no way to determine the sequence of extracellular signals and intracellular molecular events experienced by each cell in the developing tissue as it differentiates. Time-lapse imaging has been the principal go-to method to image biological processes in living systems. However, a large number of systems in biology and medicine do not permit live cell imaging methods because they are inaccessible or optically opaque, such as mouse brains and embryos. Thus, there is a crucial need to develop powerful new methods that can achieve inference of lineage information and cellular event histories from static end-point measurements. Here we propose to develop a platform that enables cells to record lineage and dynamic gene expression histories in their own genomes, within complex developing tissues. This platform will combine two recently developed tools: first, genome editing tools that can ?record? lineage information or cellular events into the genome, and second, a single molecule microscopy based in situ technology called sequential multiplexed Fluorescence In situ Hybridization (seqFISH) that can read out the recorded information in single cells without dissecting them out from tissues. We call this method MEMOIR (Memory through Enhanced Mutagenesis with Optical In situ Readout). Akin to using sequence variation for phylogenetic tree analysis, this method will allow us to reconstruct the lineage tree for a population of cells as well as the signaling event history within those cells based on the hierarchy of mutations incorporated into the target region. We have performed the proof-of-principle experiments in mouse embryonic stem cells (mESCs) and will extend the work to mouse embryos. We envision that MEMOIR will allow us to map lineages and signaling events directly in complex tissues such as the brain and track cells in metastatic tumors.

ABSTRACT A major goal in clinical neuroscience is to develop efficient treatments to prevent or minimize the loss of brain function caused by pathological decreases or increases of neuronal activity, which are hallmarks of a wide variety of neurological disorders. Interestingly, in some instances, the brain has evolved mechanisms to partially correct abnormal neuronal function. Understanding the adaptive mechanisms that restore brain function would not only provide insight into the functioning of the normal brain but also guide future approaches to ameliorate loss of brain function caused by disease or injury. We propose to start a research program to investigate the cellular and circuit mechanisms by which the brain maintains constant behavioral output, even when neuronal activity is naturally variable or it is perturbed. Our preliminary evidence with songbirds indicate that the brain circuits involved in song production demonstrate a high level of behavioral resilience both at short and long timescales. At the short timescale the patterns of firing of premotor neurons directly involved in song production vary from day to day, although there is no measurable variability in the song. At the long timescale, we genetically perturbed the activity of these premotor neurons and this caused a dramatic disruption of song. However, manipulated birds fully recovered from the perturbation, and were able to produce their original song after around 10 days. We will build on these results to explore the neuronal mechanisms that ensure behavioral resilience in a brain circuit involved in a complex behavior using gene delivery, optogenetics, in vivo functional imaging, behavioral analysis, and computational modelling.

PROJECT SUMMARY It is widely thought that identifying how neurons are connected to each other in a brain circuit, its wiring diagram, is a necessary step towards understanding how brain activity gives rise to behavior, and how it is perturbed by disease. Unfortunately, currently available methods have limitations that make it challenging to visualize these brain wiring diagrams. In addition, there is an urgent need in the field for a method that will make it possible not only to unveil brain connectivity, but also to genetically modify the functional properties of the neurons connected in a circuit. We recently developed a genetic system named TRACT and showed using Drosophila that it possesses both of these features. Unfortunately, many complex brain functions cannot be examined in Drosophila, and understanding them will require studying vertebrate animals. In recent years the zebrafish has emerged as a useful animal model to study complex brain processes, because it has a relatively simple yet conserved vertebrate brain, optical transparency during embryonic and larval stages of development, amenability to large-scale behavioral assays, the emergence of complex behaviors after only 5 days of development, and a growing suite of genetic tools that allow observation and manipulation of neuronal circuits in behaving animals. However, the usefulness of zebrafish for neuroscience research is constrained by a lack of methods to identify synaptically connected neurons. Here we propose proof-of-concept experiments to establish the TRACT system for use in zebrafish to identify the wiring diagrams of brain circuits, and to genetically manipulate the functions of neurons that mediate complex behavioral states such as sleep and arousal. These capabilities will broadly increase the usefulness of zebrafish as a model system to study vertebrate neuronal circuit function, both to reveal general principles of neuronal circuits that underlie specific behaviors, and to model complex human brain disorders such as autism, Alzheimer?s disease and schizophrenia.

PROJECT SUMMARY How does the brain balance the need to preserve prior knowledge with the necessity to continuously learn new information? The tradeo? between stability and plasticity is inherent in both biological and arti?cial learning systems constrained by ?nite resources and capacity. The hippocampus is a brain region critical for memory formation and spatial learning, which can provide a powerful experimental system for characterizing this tradeo?. The role of the hippocampus in spatial cognition is supported by the ?nding that pyramidal neurons in this area (place cells) ?re in speci?c locations in an environment (place ?elds). The population of place cells active in an environment is believed to form a neural representation or cognitive map of that environment. Spatial learning is critical for survival and involves two competing constraints: representations of space must be plastic to enable fast learning of new environments and changes in behavioral contingencies, and stable over time to enable recognition of familiar environments, reliable navigation, and leveraging of previous learning. How do these competing constraints a?ect the stability of place ?elds across time? The experimental characterization of the long-term stability of spatial representations in the hippocampus has been challenging as it requires tracking the activity of multiple place cells across extended periods of time (days to weeks). We propose to use novel approaches in large-scale electrophysiology and imaging in behaving rodents to characterize which neurons change their spatial tuning and how these changes depend on behavior. Furthermore, we will use recordings and circuit perturbations to characterize the activity patterns that predict changes in tuning stability. Our analysis will be carried out in the context of a theoretical framework for understanding the interplay between plasticity and stability of hippocampal representations. Characterizing the evolution of neural representations is of fundamental importance in understanding how information is maintained across brain circuits and how such maintenance is perturbed in brain disorders.